CN114288264B - Brain injury inflammation site trend biochemical-like nano system and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a brain injury inflammation site trend biochemical simulation nano system, a preparation method and application thereof. The invention adopts the antioxidant drug succinyl-ibuprofen and amphiphilic polymer PEG-b-PDPA, forms PP/SCB nano-particles through a film hydration method, and wraps 4T1 cell membranes on the outer layer to form the brain injury inflammation site trend biochemical MPP/SCB nano-particles; by utilizing the inflammation chemotaxis and the blood-brain barrier crossing capability of the 4T1 tumor cells, the problem that the targeting capability or the blood-brain barrier crossing capability of the brain injury inflammation part of the traditional bionic nano system is limited is solved. The bionic nano system has better treatment effect of ischemic cerebral apoplexy reperfusion injury.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a bionic nano system, and in particular relates to a brain injury inflammation part trend bionic nano system, a preparation method and application thereof.
Background
It is reported in the literature that stroke is an acute cerebrovascular disease that causes brain blood circulation to be impaired due to blockage or rupture of cerebral blood vessels, and further causes damage to brain tissues. Studies show that ischemic cerebral apoplexy accounts for about 60-80% of the total cerebral apoplexy, and the diseases have five characteristics of high morbidity, high disability rate, high mortality rate, high recurrence rate and high economic burden, and seriously affect the life quality and life health of patients. The gold standard for clinical treatment of ischemic stroke is to timely restore blood supply to ischemic brain tissue by thrombolysis or mechanical thrombolysis within a treatment time window (within 4.5 hours). Clinical practice has shown that most patients have missed optimal treatment times when admitted due to stringent thrombolytic time window limitations, at which time reperfusion injury will result if thrombolytic therapy is performed again. The pathological mechanism of reperfusion injury comprises excitotoxicity, calcium overload, oxidative stress, inflammatory response and the like, and the clinical treatment effect of the reperfusion injury is far from expected at present, so that the exploration and development of effective medicaments for treating the reperfusion injury of ischemic cerebral apoplexy is an important subject of the related field foundation and clinical research of the ischemic cerebral apoplexy.
Much research evidence suggests that oxidative stress and inflammatory responses play a major role in reperfusion injury. Reperfusion produces a large amount of active oxygen in a short period of time, inducing oxidative stress and exacerbating inflammatory reactions. Probucol is a hypolipidemic drug and has antioxidant and anti-inflammatory effects, and researches prove that the probucol has a protective effect on cerebral ischemia reperfusion injury of rats; the Succinobucol (SCB) is a mono-succinate derivative of the hypolipidemic drug probucol, has stronger antioxidant and anti-inflammatory effects than the probucol, has great potential in treating reperfusion injury caused by ischemic cerebral apoplexy, but has extremely low water solubility, cannot cross blood brain barrier to reach ischemic brain tissue, and has extremely limited clinical application.
In recent years, research shows that the slightly soluble medicine is prepared into nano particles, and the surfaces of the nano particles are wrapped by cell membranes to prepare the bionic nano system, so that the solubility of the medicine can be improved, and the medicine can be helped to cross the blood brain barrier; common cell membranes include: red blood cell membrane, neutrophil granulocyte membrane, platelet membrane, etc., while these biochemical-like nanosystems have certain targeting ability across blood brain barrier and brain injury inflammation site, but their brain injury inflammation site targeting ability or ability across blood brain barrier is limited, often need to make additional targeting modification on the surface of carrier, such as cerebral ischemia targeting peptide or T7 Tag polypeptide, etc., in order to promote more drugs to distribute to brain injury inflammation site; practice shows that the bionic nano preparation has complex components and complex preparation process. Therefore, development of a nano-preparation which has simple components and good brain injury inflammation site chemotaxis and blood brain barrier crossing ability has attracted attention of the technicians in the field.
The 4T1 cell is a breast cancer cell with high brain transfer capacity, and the surface of the cell membrane of the breast cancer cell highly expresses VCAM-1 protein and CD138 protein. The research shows that the VCAM-1 protein can be combined with VLA-4 receptors on the surfaces of a large amount of aggregated leucocytes on vascular endothelium at the brain injury inflammation part, so that 4T1 cells have good encephalitis part chemotaxis; CD138 protein can help 4T1 cells cross the blood brain barrier to reach the brain.
Based on the basis and the current situation of the prior art, the inventor of the application intends to prepare a bionic nano system by adopting 4T1 cell membrane-entrapped nano particles so as to achieve the effects of improving the aggregation of the medicine at the brain injury inflammation part and crossing the blood brain barrier; in particular to a brain injury inflammation site trend biochemical imitation nano system, a preparation method and application thereof.
Disclosure of Invention
The invention aims to provide a brain injury inflammation site trend biochemical nano system and a preparation method and application thereof based on the basis and the current situation of the prior art, and particularly relates to a simple bionic nano particle prepared by utilizing the inflammation trend and the blood brain barrier crossing capability of 4T1 tumor cells, so as to improve the aggregation of succinyl-b-the-pillar at the brain injury inflammation site and the blood brain barrier crossing effect, and realize better treatment effect of ischemic cerebral apoplexy reperfusion injury.
According to the invention, the amphiphilic high polymer copolymer PEG-b-PDPA is used for coating SCB to prepare nano-particle PP/SCB, and 4T1 cell membrane is used for coating PP/SCB to form the encephalitis trend biochemical nano-particle MPP/SCB.
Specifically, the invention adopts the antioxidant drug succinyl-ibuprofen and amphiphilic polymer PEG-b-PDPA to form PP/SCB nano-particles by a film hydration method, and 4T1 cell membranes are coated on the outer layer to form the brain injury inflammation site trend biochemical MPP/SCB nano-particles; by utilizing the inflammation chemotaxis and the blood-brain barrier crossing capability of the 4T1 tumor cells, the problem that the targeting capability or the blood-brain barrier crossing capability of the brain injury inflammation part of the traditional bionic nano system is limited is solved.
More specifically, the invention provides an MPP/SCB (brain injury inflammatory site) with a trend-like biochemical nano system.
The invention discloses a bionic nano system, which comprises a medicine, a medicine carrier and a bionic cell membrane, wherein the medicine is an antioxidant medicine for treating ischemic cerebral apoplexy; the drug carrier is an amphiphilic polymer, and the drug carrier encapsulates the drug to form a core nanoparticle by a film hydration method; the bionic cell membrane is a tumor cell membrane, and the 4T1 tumor cell membrane is used for encapsulating the kernel nanoparticles in an extrusion mode to form the brain injury inflammation site trend biochemical-like nano system.
In the invention, the antioxidant medicine for treating cerebral arterial thrombosis is preferably probucol, succinyl-ibuprofen, curcumin and baicalein, and more preferably succinyl-ibuprofen.
In the invention, the amphiphilic polymer drug carrier is polyethylene glycol-poly (isopropylamino ethyl methacrylate) (PEG-b-PDPA).
In the invention, the tumor cell membrane is: breast cancer cells (4T 1 cells) with high brain transfer capacity are lysed by using a cell lysate, the cells are broken into fragments by repeated extrusion, and finally 4T1 cell membranes are separated by using a differential centrifugation method.
The brain injury inflammation site trend imitation biochemical nano system is prepared by the following method, which comprises the following steps:
(1) Preparation of PP/SCB nanoparticles: dissolving SCB and PEG-b-PDPA in ethanol together, performing reduced pressure rotary evaporation under water bath to form a film, adding pure water, and hydrating to form PP/SCB;
(2) Preparation of MPP/SCB nanoparticles: ultrasonically mixing the 4T1 cell membrane with PP/SCB, and repeatedly extruding by an extruder to obtain MPP/SCB;
the ratio of the SCB to the PEG-b-PDPA is 2:1-1:10, and the hydration time is 30min;
the 4T1 cell membrane is extracted by the following steps: scraping 4T1 cells from the petri dish with a cell scraper and collecting into a centrifuge tube, adding membrane protein extraction reagent a (1 ml) containing 1mM protease inhibitor (PMSF), and re-suspending the cells under ice bath for 15min; repeatedly extruding the cell suspension through a polycarbonate membrane for 30 times to break the cells, centrifuging the cell debris suspension at 700g for 10min at 4 ℃, collecting supernatant to remove cell nuclei and uncleaved cells, and finally centrifuging the supernatant at 14,000g for 30min at 4 ℃ to obtain cell membrane sediment, and measuring the quality of the purified 4T1 cell membrane protein by using a BCA protein quantitative kit for later use;
in the step (2), the mass ratio of the 4T1 cell membrane (based on the contained membrane protein) to the PP/SCB is 1:1-1:10, and preferably, the mass ratio of the 4T1 cell membrane to the PP/SCB is 1:5;
in the step (2), a polycarbonate film extruder having a pore diameter of 200nm is used, and the number of times of repeated extrusion is 7 to 15, preferably 11 times.
The invention further provides application of the brain injury inflammation site trend biochemical-simulated nano system MPP/SCB in preparation of medicines for treating ischemic stroke.
According to the invention, in vitro ROS eliminating capability of MPP/SCB and protecting capability of ROS damaged cells are tested, and the results (shown in figures 2A and 2B) show that both PP/SCB and MPP/SCB can obviously eliminate ROS in PC12 cells, and compared with PP/SCB, the MPP/SCB has stronger ROS eliminating capability; the results shown in FIGS. 2C and 2D show that both PP/SCB and MPP/SCB significantly reduce apoptosis of PC12 cells caused by tBHP, and that MPP/SCB has a greater protective capacity for PC12 cells that are damaged by ROS than PP/SCB.
The MPP/SCB of the invention is measured by the in vitro blood-brain barrier crossing capability, and the quantitative detection and qualitative observation results are shown in figures 3B and 3C, compared with free DiR, the capabilities of the MPP/DiR and the MPP/DiR for crossing the blood-brain barrier are obviously improved; with the extension of the incubation time, the fluorescence in PC12 cells incubated by PP/DiR and MPP/DiR is obviously enhanced, which indicates that the amounts of PP/DiR and MPP/DiR crossing blood brain barrier are obviously increased; in addition, the fluorescence intensity in PC12 cells incubated with MPP/DiR was stronger than that in the PP/DiR group at different incubation times, indicating that MPP/DiR was stronger than PP/DiR in its ability to cross the blood brain barrier.
The in vivo distribution experimental results of the MPP/DiR of the present invention are shown in FIGS. 4A and 4C, and the free DiR group brain has no fluorescence basically, which indicates that the free DiR is difficult to cross the blood brain barrier to reach the brain; fluorescence was observed in the brain for both the PP/DiR and MPP/DiR groups, indicating that both PP/DiR and MPP/DiR can cross the blood brain barrier into the brain; in addition, the brain fluorescence intensity of the MPP/DiR group is stronger than that of the PP/DiR group, and further proves that the MPP/DiR has stronger blood-brain barrier crossing capacity; the distribution results of DiR, PP/DiR and MPP/DiR in cerebral ischemia are shown in figure 4B, the distribution of free DiR group fluorescence and PP/DiR group fluorescence in brain is not specific, and the distribution of MPP/DiR group fluorescence specificity in brain injury area proves that MPP/DiR has strong blood-brain barrier crossing capability and good inflammation tendency;
as shown in FIG. 4D, the fluorescence intensity quantitative analysis results of the main organs of each group in the invention show that the brain fluorescence intensity of the MPP/DiR group is higher than that of the DiR group and the PP/DiR group, and the ischemia half brain fluorescence intensity of the MPP/DiR group is about 7.2 times of that of the normal half brain fluorescence intensity.
The pharmacodynamics experimental results of the MPP/DiR are shown in figures 5A and 5B, and compared with a model group, the MPP/SCB and the MPP/SCB can obviously reduce the cerebral infarction area of tMCAO rats; and the MPP/SCB group had a smaller percentage of cerebral infarct size than the PP/SCB group.
The invention provides a brain injury inflammation site trend biochemical nano system, which utilizes the inflammation trend of 4T1 tumor cells and the capacity of crossing blood brain barrier to prepare simple bionic nano particles, can improve the aggregation of succinyl-bocicle at the brain injury inflammation site and the effect of crossing blood brain barrier, and can further realize better treatment effect of ischemic cerebral apoplexy reperfusion injury.
The beneficial effects obtained by the invention are as follows:
(1) The MPP/SCB of the bionic nano-system with the tendency of the brain injury inflammation part is prepared, and the problem that the bionic nano-preparation has limited inflammation tendency or blood-brain barrier crossing capacity is solved.
(2) The prepared brain injury inflammation site trend imitation biochemical nano system MPP/SCB can be used for treating ischemic cerebral apoplexy reperfusion injury, and the preparation method of the bionic nano system is simple and quick and is easy for mass production.
Drawings
FIG. 1 characterization of the trending biochemical nanosystems MPP/SCB of brain injury inflammatory sites, wherein,
(A) TEM images of PP/SCB and MPP/SCB,
(B) Particle size distribution of 4T1 cell membrane, PP/SCB and MPP/SCB,
(C) Surface protein characterization of 4T1 cells, 4T1 cell membranes, PP/SCB and MPP/SCB.
FIG. 2 in vitro ROS scavenging results for PP/SCB and MPP/SCB, wherein,
(A) Intracellular ROS inverted fluorescence plots, scale bar, following incubation of PP/SCB and MPP/SCB with t-butylhydroperoxide (tBHP) -treated PC12 cells, respectively: the particle size of the particles is 50 μm,
(B) Intracellular ROS horizontal flow quantitative determination results after PP/SCB and MPP/SCB are respectively incubated with PC12 cells treated by tBHP,
(C) Apoptosis flow assay results after PP/SCB and MPP/SCB were incubated with tBHP treated PC12 cells, respectively,
(D) Cell viability assay results after PP/SCB and MPP/SCB were incubated with tBHP treated PC12 cells, respectively.
FIG. 3PP/DiR and MPP/DiR in vitro cross blood brain barrier results, wherein,
(A) Constructing a Transwell experimental schematic diagram of a blood brain barrier model in vitro,
(B) The average fluorescence intensity of PC12 cells uptake under different treatment modes was determined,
(C) Laser confocal images taken by PC12 cells under different processing modes, scale: 25 μm.
Fig. 4 results of in vivo distribution of MPP/DiR, wherein,
(A) Representative fluorescence imaging of major organs of ischemia reperfusion (tMCAO) rat model 24h after intravenous injection of different formulations,
(B) Representative fluorescence imaging of brain sections of tMCAO rats 24h after intravenous injection of the different formulations,
(C) Representative fluorescence imaging of tMCAO rat brain 24h after intravenous injection of the different formulations,
(D) Fluorescence quantification of major organs and brain in tMCAO rats 24h after intravenous injection of the different formulations.
Fig. 5, in vivo pharmacodynamic diagram of MPP/SCB, wherein,
(A) Representative images of TTC staining of rat brain sections 24h after intravenous injection of the different formulations,
(B) Quantitative results of the percentage of cerebral ischemic area in rats 24h after intravenous injection of the different formulations.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, components, and/or combinations thereof.
As described in the background art, the existing bionic nano system has the problem of limited targeting ability of a brain injury inflammation part or limited blood-brain barrier crossing ability, and in order to solve the problem, the invention provides an MPP/SCB of the brain injury inflammation part trend bionic nano system, which consists of SCB, amphiphilic polymer PEG-b-PDPA and 4T1 cell membrane.
The invention provides a preparation method of an MPP/SCB (MPP/SCB) imitating a biochemical nano system with tendencies of brain injury inflammation parts, which comprises the following steps:
(1) Preparation of PP/SCB nanoparticles: SCB and PEG-b-PDPA are dissolved in ethanol together, and are subjected to reduced pressure rotary evaporation under water bath to form a film, and then are added with pure water and hydrated for 30min to form PP/SCB.
Preferably, the mass ratio of SCB to PEG-b-PDPA in the step (1) is 1:3-10.
(2) Preparation of MPP/SCB nanoparticles: mixing 4T1 cell membrane with PP/SCB, repeatedly squeezing to obtain MPP/SCB.
Preferably, the specific steps of the MPP/SCB preparation in the step (2) are as follows:
s1, scraping and collecting 4T1 cells from a culture dish by adopting a cell scraper, adopting a cell membrane extraction kit, cracking the cells by a repeated extrusion method, and separating and collecting the cells by a differential centrifugation method to obtain the 4T1 cell membrane.
S2, ultrasonically mixing the 4T1 cell membrane with the PP/SCB, repeatedly extruding through a 200nm polycarbonate membrane, and fully fusing the PP/SCB nanoparticle with the 4T1 cell membrane to obtain the encephalitis trend biochemical MPP/SCB nanoparticle.
Preferably, in the step S2, the mass ratio of the 4T1 cell membrane (based on the amount of the contained membrane protein) to PP/SCB is 1:5-10.
Preferably, the number of repeated pressing in the step S2 is 7 to 15.
The invention also provides application of the brain injury inflammatory part trend biochemical-simulated nano system MPP/SCB in treatment of ischemic cerebral apoplexy reperfusion injury.
The invention will be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not to be construed as limiting the invention. If experimental details are not specified in the examples, it is usually in accordance with conventional conditions, or in accordance with the recommended conditions of the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 preparation of MPP/SCB
(1) And precisely weighing 6mg of PEG-b-PDPA and 2mg of SCB into an eggplant-shaped bottle by an analytical balance, adding absolute ethyl alcohol for ultrasonic dissolution, performing reduced pressure rotary evaporation at 45 ℃ to form a film, and then adding 5ml of deionized water for hydration for 30min to obtain PP/SCB nanoparticles.
(2) 4T1 cells were scraped from the petri dish using a cell scraper and collected into a centrifuge tube, membrane protein extraction reagent A (1 ml) containing 1mM protease inhibitor (PMSF) was added, and the cells were resuspended in an ice bath for 15min. The cell suspension was then repeatedly extruded through a polycarbonate membrane 30 times to disrupt the cells. The cell debris suspension was then centrifuged at 700g for 10min at 4℃and the supernatant was collected to remove nuclei and uncleaved cells. Finally, the supernatant was centrifuged at 14,000g for 30min at 4℃to obtain a cell membrane pellet. The purified 4T1 cell membrane protein was quantified for subsequent use using BCA protein quantification kit.
(3) Taking 5mg of PP/SCB nanoparticles and 1mg of 4T1 cell membrane, carrying out ultrasonic treatment to uniformly mix, and repeatedly extruding through a 200nm polycarbonate membrane for 11 times to obtain 4T1 cell membrane coated PP/SCB nanoparticles MPP/SCB.
Example 2 characterization of physicochemical Properties of MPP/SCB
(1) One drop of PP/SCB or MPP/SCB solution (1 mg/ml) is dropped on a carbon film copper net, and after natural drying, one drop of phosphotungstic acid solution is dropped for dyeing for 2-3min, and then a transmission electron microscope is used for observing the morphology of the two nano particles. As shown in FIGS. 1A and 1B, the PP/SCB and the MPP/SCB are spherical with round particle size, the MPP/SCB has an obvious membrane shell structure, and the thickness of the outer membrane is about 20nm.
(2) The particle sizes of the PP/SCB and MPP/SCB solutions are tested by a laser particle size analyzer, the particle size result is shown in figure 1B, the particle size of the PP/SCB is 54.1+/-1.6 nm, and the polydispersity index is 0.22+/-0.01; the particle size of MPP/SCB was 67.8.+ -. 0.6nm, and the polydispersity index was 0.29.+ -. 0.01. The particle size result is consistent with an electron microscope photograph, and further shows that the biochemical imitation MPP/SCB nano-particle is successfully prepared.
(3) The surface proteins of 4T1 cells, 4T1 cell membranes, PP/SCB and MPP/SCB are detected by using a western blot technique. As shown in FIG. 1C, the CD47, CD138 and VCAM-1 proteins were detected on the 4T1 cell, 4T1 cell membrane and MPP/SCB surface, while no protein was detected on the PP/SCB surface, indicating that the protein integrity of the 4T1 cell membrane surface was not destroyed during cell membrane extraction and MPP/SCB preparation.
Example 3 in vitro ROS-eliminating ability of MPP/SCB and protection ability against ROS-damaged cells
(1) PC12 cell reperfusion injury model was constructed by incubating with t-butyl hydroperoxide solution (tBHP, 100. Mu.M) and PC12 cells for 1 h. The PC12 cells were then incubated with DMEM medium containing PP/SCB and DMEM medium containing MPP/SCB, respectively, for 4h. Then adding a cell ROS probe DCFH-DA, and respectively qualitatively and quantitatively detecting the level of ROS in each group of cells by using a fluorescence inversion microscope and a flow cytometer; as shown in FIGS. 2A and 2B, both PP/SCB and MPP/SCB significantly eliminate ROS within PC12 cells, and MPP/SCB has a greater ability to eliminate ROS than PP/SCB.
(2) PC12 cell reperfusion injury model was constructed by incubating with t-butyl hydroperoxide solution (tBHP, 100. Mu.M) and PC12 cells for 1 h. The PC12 cells were then incubated with DMEM medium containing PP/SCB and DMEM medium containing MPP/SCB, respectively, for 4h. And detecting apoptosis and necrosis of each group of cells by using an Annexin V-FITC/PI apoptosis kit. As shown in FIGS. 2C and 2D, both PP/SCB and MPP/SCB significantly reduced apoptosis of PC12 cells caused by tBHP, and MPP/SCB showed a stronger protective ability against PC12 cells suffering from ROS damage than PP/SCB.
Example 4 in vitro measurement of MPP/SCB ability to cross the blood brain Barrier
And respectively preparing the PP/DiR nanoparticle and the MPP/DiR nanoparticle by replacing the SCB with the fluorescent dye DiR. By using the transwell plate construct outer blood brain barrier model, as shown in FIG. 3A, mice brain microvascular endothelial cells (bEnd.3 cells) were inoculated in the upper chamber of the transwell plate, and cultured until the transmembrane resistance reached 330ohm cm 2 PC12 cells were then seeded under the transwell plates. After cell attachment, the upper cells were filled with free fluorescent dye DiR, PP/DiR or MPP/DiR (corresponding to 10. Mu.g/ml DiR, respectively). After culturing for 12h and 24h respectively, detecting the fluorescence intensity in PC12 cells at the lower layer of the transwell plate by using a flow cytometer; cells were fixed with 4% paraformaldehyde, nuclei of PC12 cells were stained with 5ug/ml DAPI staining solution, and uptake of each group of PC12 cells was observed with a laser confocal microscope. The quantitative detection and qualitative observation results are shown in figures 3B and 3C, and compared with free DiR, the PP/DiR and MPP/DiR have obviously improved blood brain barrier crossing capacity; with the extension of the incubation time, the fluorescence in PC12 cells incubated by PP/DiR and MPP/DiR is obviously enhanced, which indicates that the amounts of PP/DiR and MPP/DiR crossing blood brain barrier are obviously increased; in addition, the fluorescence intensity in PC12 cells incubated with MPP/DiR was stronger than that in the PP/DiR group at different incubation times, indicating that MPP/DiR was stronger than PP/DiR in its ability to cross the blood brain barrier.
Example 5 in vivo distribution experiment of MPP/DiR
Rat tMCAO model was established by the wire-plug method. Male SD rats (250-280 g) were fasted for 12h before surgery and anesthetized by intraperitoneal injection of 1% pentobarbital at a dose of 40 mg/kg. After disinfecting the skin of the rat with iodophor, cutting off the skin of the neck, and sequentially separating the left Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA), wherein the three arteries are distributed in a Y shape. Since the common carotid artery and the vagus nerve are concomitant, care should be taken not to disrupt the vagus nerve when isolating the common carotid artery. Then, the CCA and ICA are clamped, the distal end of the ECA is ligated, a V-shaped small opening is cut at the proximal end of the ECA by using ophthalmology, a nylon wire plug is inserted from the small opening, the Y-shaped bifurcation is turned to the ICA, an artery clamp at the ICA is opened, and the wire plug is pushed inwards slowly by about 18mm to block the blood supply of middle cerebral artery. To prevent the plug from slipping off, the plug was secured with a suture at the proximal end of the ECA, from which time cerebral ischemia was recorded. The arterial clamp at CCA was opened and the rat skin tissue was sutured. The plug was slowly withdrawn 2h after the plug and the black spot mark on the plug was exposed to the outside to effect reperfusion. The rats were simultaneously injected with free DiR, PP/DiR and MPP/DiR, respectively, via the tail vein (corresponding to 0.1mg/kg of DiR, respectively).
(1) Immediately after 22h of dosing, rats were sacrificed, the brains were broken and the main organs were removed, and the distribution of DiR, PP/DiR and MPP/DiR in the brains and main organs was observed with a small animal biopsy imager. The experimental results are shown in figures 4A and 4C, where the free DiR group brains are essentially non-fluorescent, indicating that free DiR is difficult to cross the blood brain barrier into the brain; fluorescence was observed in the brain for both the PP/DiR and MPP/DiR groups, indicating that both PP/DiR and MPP/DiR can cross the blood brain barrier into the brain; in addition, the MPP/DiR group has stronger brain fluorescence intensity than the PP/DiR group, and further proves that the MPP/DiR has stronger blood-brain barrier crossing capability.
(2) The brain was cut into 2mm thick continuous slices along the coronal plane, the distribution of DiR, PP/DiR and MPP/DiR in the brain was observed with a small animal biopsy imager, and the brain slices were stained with TTC staining solution, which stained normal brain tissue red and ischemia white. The distribution of DiR, PP/DiR and MPP/DiR at cerebral ischemic sites was observed. The experimental results are shown in fig. 4B, the distribution of free DiR group fluorescence and PP/DiR group fluorescence in brain is not specific, and the distribution of MPP/DiR group fluorescence specificity in brain injury area proves that MPP/DiR has strong blood-brain barrier crossing capability and good inflammation orientation.
(3) The fluorescence intensity of each group of main organs was quantitatively analyzed, and the experimental results are shown in fig. 4D, wherein the brain fluorescence intensity of the MPP/DiR group is higher than that of DiR group and PP/DiR group, and the ischemic cerebellum fluorescence intensity of the MPP/DiR group is about 7.2 times that of the normal cerebellum fluorescence intensity.
Example 6 pharmacodynamic experiments with MPP/DiR
A rat tMCAO model was constructed as in example 3, and the plugs were withdrawn 2h after embolization to effect reperfusion. Simultaneously, rats were injected with PP/SCB and MPP/SCB (corresponding to 10mg/kg SCB, respectively) via the tail vein. Immediately after 22h of dosing, the rats were sacrificed and brains were removed, the brains were cut into 2mm thick continuous sections along the coronal plane, the brain sections were stained with TTC staining solution, the improvement of ischemic brain tissue by PP/SCB and MPP/SCB was observed, and the percentage of ischemic area for each group was quantitatively calculated. As shown in the experimental results in figures 5A and 5B, compared with the model group, the PP/SCB and the MPP/SCB can obviously reduce the cerebral infarction area of tMCAO rats; and the MPP/SCB group had a smaller percentage of cerebral infarct size than the PP/SCB group.
In conclusion, the invention prepares the bionic nano system MPP/SCB for the trend of the brain injury inflammation part for the first time, and solves the problem that the bionic nano preparation has limited inflammation trend or blood brain barrier crossing capability. And the MPP/SCB has good curative effect on ischemic cerebral apoplexy reperfusion injury.
Claims (8)
1. The bionic nano system for treating the trend of the cerebral injury inflammation part is characterized by comprising a medicine, a medicine carrier and a bionic cell membrane, wherein the medicine is an antioxidant medicine for treating ischemic cerebral apoplexy; the drug carrier is an amphiphilic polymer, and the drug carrier encapsulates the drug to form a core nanoparticle by a film hydration method; the bionic cell membrane is a tumor cell membrane, and the tumor cell membrane encapsulates the kernel nanoparticle in an extrusion mode to form a brain injury inflammation site trend biochemical-like nano system;
the antioxidant medicine for treating cerebral arterial thrombosis is selected from probucol, succinyl-ibuprofen, curcumin, and other drugs,
Baicalein;
the amphiphilic polymer drug carrier is polyethylene glycol-poly (isopropylamino ethyl methacrylate)
PEG-b-PDPA；
The tumor cell membrane is: breast cancer cell 4T1 cells with brain high transfer capacity are adopted
Cell lysate is used to lyse cells, and the cells are broken into pieces by repeated extrusion, and finally differential centrifugation is used
The method is used for separating and obtaining the 4T1 cell membrane.
2. The brain injury inflammatory site trend biochemical nano-system according to claim 1, wherein,
the antioxidant medicine for treating cerebral arterial thrombosis is selected from succinyl-ibuprofen.
3. The brain injury inflammatory site trend-imitating biochemical nano system according to claim 1, wherein the brain injury inflammatory site trend-imitating biochemical nano system is prepared by the following method:
(1) SCB and PEG-bPDPA is dissolved in ethanol together, and is decompressed and distilled under water bath to form a film,
then adding pure water and hydrating to form PP/SCB;
(2) And (3) ultrasonically mixing the 4T1 cell membrane with the PP/SCB, and repeatedly extruding through an extruder to obtain the MPP/SCB.
4. The brain injury inflammatory site trend biochemical nano system according to claim 3, wherein,
in the step (1) of the preparation method, SCB and PEG-bThe mass ratio of the PDPA is 2:1-1:10, and the hydration time is 30 min.
5. The brain injury inflammatory site trend biochemical nano system according to claim 3, wherein,
in the preparation method step (2), the extraction steps of the 4T1 cell membrane are as follows: scraping 4T1 cells from a culture dish by adopting a cell scraper, collecting the cells into a centrifuge tube, adding a membrane protein extraction reagent A1 ml containing A1 mM protease inhibitor PMSF, and re-suspending the cells under an ice bath for 15min; repeatedly extruding the cell suspension through a polycarbonate membrane for 30 times to break the cells; the cell debris suspension was then centrifuged at 700g for 10min at 4 ℃ and the supernatant was collected to remove nuclei and uncleaved cells; finally, the supernatant was centrifuged at 14,000, 000g for 30min at 4 ℃ to obtain a cell membrane pellet; the quality of the purified 4T1 cell membrane protein is determined by using a BCA protein quantification kit for standby.
6. The brain injury inflammatory site trend biochemical nano system according to claim 3, wherein,
in the preparation method step (2), the mass ratio of the 4T1 cell membrane to the PP/SCB is 1:1-1:10 based on the contained membrane protein.
7. The brain injury inflammatory site trend biochemical nano system according to claim 6, wherein,
in the preparation method step (2), the mass ratio of the 4T1 cell membrane to the PP/SCB is 1:5.
8. The brain injury inflammatory site trend biochemical nano system according to claim 3, wherein,
in the step (2) of the preparation method, the extruder is a polycarbonate membrane extruder with the aperture of 200-nm, and the number of times of repeated extrusion is 7-15 times.
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